Methods and compositions for detecting single T cell receptor affinity and sequence
Provided herein are methods for determining the T cell receptor affinity and sequence of antigen-specific T cells using a micropipette adhesion assay and single cell paired TCRα/TCRβ sequencing. Further provided are methods for the treatment of viral infections or cancers by adoptive transfer of high affinity functional T cells.
1. A method for measuring T cell receptor (TCR) affinity and obtaining TCR sequence comprising:
(a) obtaining a population of antigen-specific T cells from a starting population of T cells using pMHC-tagged streptamers;
(b) individually performing a micropipette adhesion assay on each of said antigen-specific T cells, thereby measuring the two-dimensional (2D) TCR affinity to a peptide-major histocompatibility complex (pMHC); and
(c) sequencing the TCR of each of said antigen-specific T cells by paired TCRα/TCRβ sequencing, thereby obtaining the sequence of said TCR.
2. The method of claim 1 , wherein antigen-specific T cell isolation comprises:
(a) staining the starting population of T cells with pMHC-tagged streptamers;
(b) sorting for the streptamer-bound T cells; and
(c) dissociating the streptamers from the T cells, thereby obtaining a population of antigen-specific T cells.
3. The method of claim 2 , wherein dissociating comprises incubating the streptamer-bound T cells in a dissociation buffer comprising biotin and/or sodium azide.
4. The method of claim 3 , wherein the streptamer-bound T cells are incubated for about 15 minutes to about 90 minutes.
5. The method of claim 2 , wherein antigen-specific T cell isolation is at 3° C. to 5° C.
6. The method of claim 1 , wherein step (a) comprises in vitro clonal expansion of an antigen-specific T cell by co-culturing a population of polyclonal T cells with antigen presenting cells loaded with antigen peptide and/or activating a population of polyclonal T cells with CD3 and CD28.
7. The method of claim 1 , wherein the starting population of T cells is a peripheral blood sample comprising CD8 + cytotoxic T Lymphocytes (CTLs) or CD4 + T cells obtained from a patient sample or healthy blood donor sample.
8. The method of claim 1 , wherein the population of antigen-specific T cells comprises less than 200 T cells or less than 100 T cells.
9. The method of claim 1 , wherein the antigen is a tumor associated self-antigen, tumor neo-antigen, or viral antigen.
10. The method of claim 9 , wherein the viral antigen is HCV.
11. The method of claim 1 , wherein step (b) comprises contacting the antigen-specific T cells with red blood cells coated with agonist pMHC and measuring adhesion frequency.
12. The method of claim 11 , wherein the contacting is for 2 seconds to 6 seconds or 3 seconds to 5 seconds.
13. The method of claim 11 , further comprising measuring the TCR and pMHC site density and calculating 2D affinity from adhesion probability and site density.
14. The method of claim 13 , further comprising estimating that variance associated with the 2D TCR affinity measurement comprising:
(a) measuring the standard deviation of TCR site density fluctuation between T cells by measuring the adhesion frequencies of one red blood cell (RBC) coated with anti-TCR antibody against multiple T cells;
(b) measuring the standard deviation of pMHC site density fluctuation between RBCs by measuring the adhesion frequencies of one antigen-specific T cell against multiple RBCs coated with pMHC; and
(c) measuring the standard deviation of TCR site density fluctuation on the surface of one T cell by measuring the adhesion frequencies of one RBC coated with anti-TCR antibody against multiple areas of one T cell.
15. The method of claim 11 , wherein the adhesion frequency is between 30% and 80%.
16. The method of claim 1 , wherein step (c) comprises transferring the antigen-specific T cells individually by micropipette into lysis buffer for reverse transcription, PCR amplification of TCRα/TCRβ, and next-generation sequencing.
17. The method of claim 1 , wherein the paired TCRα/TCRβ sequencing is performed on single antigen-specific T cells.
18. The method of claim 16 , further comprising separating reads according to cell barcodes and clustering reads based on sequence similarity.
19. The method of claim 1 , wherein the TCR affinity is obtained and TCR sequence is amplified in 15 hours to 48 hours or 20 hours to 24 hours.
20. A method of monitoring the TCR repertoire of a subject comprising obtaining TCR affinity and sequence according to claim 1 .
21. The method of claim 16 , wherein the PCR amplification of TCRα/TCRβ comprises primers sequences selected from Table 1 and/or Table 2.
22. The method of claim 1 , wherein the antigen is an antigen associated with a disease.